The present invention relates to enzyme-containing polyurethanes, and, especially, to enzyme-containing polyurethanes of relatively high enzyme loading and relatively high catalytic (enzyme) activity.
It has been known for some time that one can incorporate proteins within polyurethane polymers during polymer synthesis. For example, U.S. Pat. Nos. 3,928,138, 3,929,574, 4,098,465, 4,195,127, and 4,250,267 describe enzymes bound within a hydrophilic polyurethane polymer. Although enzyme activity was evident in those polymers, no attempt was made to quantify the degree of enzyme activity within the polymers.
Academics have more recently begun to revisit the synthesis of enzymatic polyurethane. For example, Dias et al. assessed the performance of lipase incorporated within polyurethane foams. Dias, S. F., Vilas-Boas, L., Cabral, J. M. S., and Fonseca, M. M. R., Biocatalysis, 5, 21 (1991). That study described the synthesis of enzymatic polymers without the use of additives, enzyme stabilizers, or enzyme pre-modification. Enzyme concentration within the polymers was varied over a broad range in the course of this study. Those studies indicated an apparent reduction in enzyme activity retention at high enzyme loading (for example, greater than 0.1 weight percent).
Storey et al described the immobilization of amyloglucosidase enzyme within several types of crosslinked polyurethane matrices Storey, K. B., Duncan, J. A., Chakrabarti, J. A., Appl. Biochem. Biotechnol. 23, 221 (1990). The enzyme concentrations employed in that study were relatively dilute and the use of additives or other non-essential components was not explored.
Recent studies of general polyurethane synthesis (irrespective of incorporation of enzyme therein) have shown that incorporation of a surfactant in the reaction mixture can lead to desirable physical properties of the polyurethane polymer product. It is believed that surfactants stabilize the carbon dioxide bubbles that are formed during synthesis and are responsible for foaming. For example, certain surfactants have been found to promote the creation of small carbon dioxide bubble, resulting in formation of a polymer product having a morphology similar to a fabric. Other surfactant have been found to promote relatively large carbon dioxide bubbles, resulting in a polymer product having a morphology similar to a sponge. Given the control that surfactants enable over the physical/morphological characteristics of polyurethanes, suppliers of polyurethane prepolymer typically recommend that surfactant be added to a polyurethane reaction mixture.
Thus, recent studies of the synthesis of enzyme-containing polyurethanes have employed surfactants to alter/control the physical properties of the resultant polymers. For example, a number of studies describe the immobilization of organophosphorus hydrolase using a polyurethane polymer synthesis strategy in which a variety of non-ionic surfactants were used as additives to alter the physical properties polymers. Havens, P. L., Rase, H. F., Ind. Eng. Chem. Res., 32, 2254 (1993); LeJeune, K. E., Swers, J. S., Hetro, A. D., et al. Biotechnol. Bioeng., 64, 2, 250 (1999); Lejeune, K. E., et al. Biotechnol. Bioeng., 54, 105, (1997); LeJeune, K. E. and Russell, A. J. Biotechnol. Bioeng., 51, 450 (1996). In general, these surfactants were used in an attempt to optimize the performance of the polyurethane sponge product in a particular application. For example, the studies of Havens and Rase were focused upon using the resultant polymers as column packing material and as adsorbent sponges to decontaminate pesticide spills. The studies reported varying surfactant hydrophobicity could produce polymers that were better suited for a particular application. The enzyme concentration/loading employed in the studies of Havens and Rase and the other studies was quite low (in general, well below 0.1 weight percent of the polymer).
It is desirable to develop enzyme containing polymers and methods of synthesis of such polymers in which enzyme loading and enzyme activity are improved.
The present inventors have discovered that certain surfactants not only enable control of polyurethane physical properties/morphology, but enhance the activity of immobilized enzymes at relatively high enzyme loading. As used herein, the term xe2x80x9cenzymexe2x80x9d refers to a protein that catalyzes at least one biochemical reaction. A compound for which a particular enzyme catalyzes a reaction is typically referred to as a xe2x80x9csubstratexe2x80x9d of the enzyme. Enzymes typically have molecular weights in excess of 5000.
In general, six classes or types of enzymes (as classified by the type of reaction that is catalyzed) are recognized. Enzymes catalyzing reduction/oxidation or redox reactions are referred to generally as EC 1 (Enzyme Class 1) Oxidoreductases. Enzymes catalyzing the transfer of specific radicals or groups are referred to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis are referred to generally as EC 3 hydrolases. Enzymes catalyzing removal from or addition to a substrate of specific chemical groups are referred to generally as EC 4 Lyases. Enzymes catalyzing isomeration are referred to generally as EC 5 Isomerases. Enzymes catalyzing combination or binding together of substrate units are referred to generally as EC 6 Ligases.
In one aspect, the present invention provides a method of increasing loading of active enzyme immobilized in a polyurethane polymer including the steps of:
synthesizing the polyurethane polymer in a reaction mixture containing water and enzyme; and
including a sufficient amount of a surfactant in the reaction mixture to increase enzyme activity at an enzyme loading (as compared to a polymer of the same enzyme loading synthesized without surfactant).
As used herein, the term xe2x80x9csurfactant: refers generally to a surface active agent that is reduces the surface tension of a liquid (water, for example) in which it is dissolved.
Preferably, the surfactant is nonionic and comprises between 0.5 to 5.0 weight percent of the aqueous component of the mixture. In the synthesis of the polyurethanes of the present invention, urethane prepolymers were mixed with water. The aqueous component of the reaction mixture included water, enzyme, surfactant and buffer salts. The weight percent surfactant in the aqueous component is thus calculated by dividing the weight of the surfactant by the weight of the entire aqueous component and multiplying the result by 100%. The enzyme loading in the present invention can be greater than approximately 0.1 percent by weight of the polyurethane polymer (weight of enzyme/[weight of enzyme-containing polymer product]*100%) while retaining substantial enzyme activity. Relatively high activity is maintained even when the enzyme loading is greater that approximately 0.5 percent by weight of the polyurethane polymer. Indeed, relatively high activity is maintained even when the enzyme loading is greater that approximately 1 percent by weight of the polyurethane polymer.
The polyurethane polymers of the present invention preferably include at least one of an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase. Examples of enzymes suitable for use in the present invention include, but are not limited to, a lipase, a peroxidase, a tyrosinase, a glycosidase, a nuclease, a aldolase, a phosphatase, a sulfatase, or a dehydrogenase.
More than one type of enzyme are easily co-immobilized within the polyurethane polymer. The enzymes can be within the same class (for example, two hydrolases) or a within different classes of enzyme.
In another aspect, the present invention provides a polyurethane polymer containing an enzyme loading of more than approximately 0.1 weight percent. The polyurethane polymer is synthesized in the presence of a sufficient amount of a surfactant (preferably, nonionic) to increase enzyme activity at the enzyme loading of the polymer (as compared to the case when no surfactant is used).
In still another aspect, the present invention provides a method of improving enzymatic activity of a polyurethane polymer synthesized with an enzyme loading of more than approximately 0.1 weight percent. The method includes the step of:
adding a sufficient amount of a surfactant (preferably, nonionic) to during synthesis of the polyurethane polymer to increase enzyme activity at the enzyme loading.
The polymers and methods of the present invention provide enhanced enzyme activity retention as the enzyme loading or enzyme content of such polymers is increased (for example, to above approximately 0.1 weight percent of the polymer). Relatively large quantities of enzymes are immobilized within the polymers of the present invention while retaining a significant portion of the native enzyme specific activity.